Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

Characterization of subcellular fractions and distribution profiles of transport components involved in Ca(2+) homeostasis in rat vas deferens.

INTRODUCTION: The sarcoplasmic reticulum present in eukaryotic cells contains Ca(2+) pumps (SERCA type) that accumulate Ca(2+) from the cytosol and Ca(2+) channels, such as ryanodine receptors and inositol 1,4,5-trisphosphate receptors, that release Ca(2+) from the lumen of this organelle. The use of a preparation rich in sarcoplasmic reticulum vesicles and poorly contaminated with plasmalemmal vesicles would be a prerequisite for studies of Ca(2+) efflux through ryanodine and inositol 1,4,5-trisphosphate receptors, so the present work was aimed to characterize the distribution profiles of various markers of sarcoplasmic reticulum and plasma membrane among fractions obtained from rat vas deferens. METHODS: Oxalate-dependent Ca(2+) uptake, thapsigargin-sensitive (Ca(2+)-Mg(2+)) ATPase activity and binding of [3H]ryanodine and [3H]inositol 1,4,5-trisphosphate were measured in the nuclear, mitochondrial, and microsomal fractions obtained by differential centrifugation of rat vas deferens homogenate. RESULTS: The recovery of the thapsigargin-resistant (Ca(2+)-Mg(2+)) ATPase activity, supposed to label the plasma membrane, was the same among nuclear, mitochondrial, and microsomal fractions, whereas the recovery of the thapsigargin-sensitive (Ca(2+)-Mg(2+)) activity, oxalate-dependent Ca(2+) uptake, and [3H]inositol 1,4,5-trisphosphate binding, used as sarcoplasmic reticulum markers, was higher in nuclear fraction than in the others. The recovery profiles of the four sarcoplasmic reticulum markers, including [3H]ryanodine binding, were statistically the same among the different subcellular fractions. Caffeine, an agonist of ryanodine receptors, induced the release of 17% of Ca(2+) taken up by the vesicles present in the nuclear fraction but had no effect in microsomes. DISCUSSION: Although this nuclear fraction is less purified in sarcoplasmic reticulum markers than the microsomal fraction, it is more suitable for studying Ca(2+) release through ryanodine receptors, primarily because it is less contaminated with vesicles from the plasma membrane which are able to take up Ca(2+) but are insensitive to caffeine.